The total quantity of biomass and its growth rate are key parameters in most bioprocesses. Both the measurement technology and method depend on the culture, the vessel, and the type of organism.
There are number of key elements to consider when choosing a device for biomass measurement. These include:
Two optical technologies are widely used to estimate biomass within a bioprocess: Spectrophotometry measured at a wavelength of 600 nm (commonly abbreviated as OD600) and backscattered light measurement. Backscattering light measurement allows for a more sophisticated monitoring process, the data is available immediately and changes in the culture can be observed in real-time. OD biomass measurement is performed offline, which may be easier to use when starting out. Deciding which one is the best for your process requires the knowledge of both working principles and how they influence the measurement.
Measuring the optical density is a well-known, long time practiced offline technique, used and described in many publications. You need a spectrophotometer, which is commonly available in the laboratory. For a biomass measurement a culture sample is taken from the cultivation vessel and deposited into a cuvette. The cuvette is placed in the spectrophotometer inside a light beam with a fixed wavelength (usually 600 nm). The device measures the relative light intensity before and after the sample. Using the Lambert Beer equation, the difference between these values can be used to calculate the optical density of the culture.
Benefits:
However, the drawbacks are:
A device comprising of a light source and a light detector comparable to the optical density is required for the backscattered light method. In contrast to the OD, the light source and detector are located on the same side of the sample. Light is emitted from the source and into the culture liquid. The photons interact with the cells and are scattered in different directions. Some photons return to the photodiode detector, converting the scattered light intensity into an electric current. Higher cell densities increase the probability of an incident photon interacting with a cell. With increasing cell density, more light is scattered back to the photodiode.
Backscatter light method. Source: aquila biolabs 2018
Benefits
Continuous measurement of the filamentous organism Aspergillus niger using backscattered light. Source: aquila biolabs 2019
The drawbacks are:
Both OD and backscatter are adequate methods of estimating the biomass in shake flasks. While OD is able to precisely measure lower cell densities and more common values, backscattering eliminates the need for dilutions and can be used with very high cell densities. Therefore, both technologies can be used equally as well when comparing external devices, depending on the focus of the cultivation.
The biggest differences can be seen when comparing the methods for taking measurements. Using an automated online system the data is available immediately and changes in the culture can be observed in real-time. This decreases the response time and prevents the researcher from sampling when nothing “interesting” happens. Online biomass measurement also provides a complete characterization of the culture, and there are no down-times on weekends or evenings, as there would be with offline measurements.
As seen in many other industrial fields, process automation is often desired as it saves time and eliminates human error. This will also mean purchasing additional equipment, which is where weighing up the pros and cons will influence your decision.
Bernd Leuchtle studied biology with a focus on biotechnology and biochemical engineering in Aachen, Germany. After acquiring his doctorate and a period as post doc, he joined the team of aquila biolabs as an application specialist.
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